Our long-term goal is to isolate and chemically charaterize each protein product arising from the seventen nif (nitrogen fixation) genes of the free-living (non-symbiotic) nitrogen fixing organism Klebsiella penumoniae. Furthermore, we plan to determine the enzymatic function of the gene products which convert precursor peptides into the enzyme nitrogenase, including those which synthesize the molybdenum-iron cofactor from molybdate in the growth medium. We plan to determine the mechanism of action of the gene products which oxidize pyruvate and pass electrons to nitrogenase. Ultimately, we hope to crystallize for structral characterization each of these seventeen proteins. The fundamental bio-chemistry of this complex multigene system seems likely to invlove novel features. From the proposed project period (1987-92), we plan to (a) determine the way in which nifM product converts nifH product into the iron protein of nitrogenase; (b) determine the mode of maturation of nifK and nifD into the MoFe protein component of nitrogenase; (c) determine the pathway of MoFe confactor biosynthesis; and (d) determine the mechanism by which pyruvate s oxidized by nifJ. The methods used include combinatins of nif genes, large-scale (1000L) culture of E. coli. harboring nif-bearing plasmids, and examination of the purified gene products for enzymic function under rigorous anaerobiosis.